EP2470164B1 - Zusammensetzungen enthaltend liposomen und deren verwendung - Google Patents

Zusammensetzungen enthaltend liposomen und deren verwendung Download PDF

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EP2470164B1
EP2470164B1 EP10761069.3A EP10761069A EP2470164B1 EP 2470164 B1 EP2470164 B1 EP 2470164B1 EP 10761069 A EP10761069 A EP 10761069A EP 2470164 B1 EP2470164 B1 EP 2470164B1
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protein
cells
cell
liposomes
composition
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EP2470164A2 (de
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Marcelle Machluf
Tomer Bronshtein
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Technion Research and Development Foundation Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention in some embodiments thereof, relates to liposomal compositions.
  • Liposome based DNA and drug delivery systems have been extensively investigated in the last four decades, and used as a mean to treat various conditions. Liposomal systems allow the efficient entrapment of both hydrophilic and hydrophobic compounds in a well-characterized, biocompatible and non-immunogenic lipid vesicle that can range from nanometers to micrometers in diameter. Liposomes can also be targeted using specific ligands such as protein conjugates or antibodies that bind specific cellular receptors. In cancer therapy, liposomal systems are of the most popular and well-investigated drug carriers. This is mainly due to the enhanced permeability and retention (EPR) effect, which refers to the increased vascular permeability of tumor vessels due to tumor angiogenesis.
  • EPR enhanced permeability and retention
  • the EPR effect results in the accumulation of liposomes in the tumor extracellular fluid, which is exploited as a passive targeting mechanism.
  • State of the art technologies in liposomal drug delivery for cancer therapy primarily include drugs that are approved for clinical use (e.g., DaunoXomeTM, MyocetTM, DoxilTM, CaelyxTM).
  • drugs that are approved for clinical use
  • Several approaches are currently investigated for the targeting of liposomal systems to cancer, which include the binding of targeting moieties to the liposome surface (e.g., antibodies).
  • Synthetic cationic liposomes are the most common vectors for DNA delivery although their cytotoxicity remains a concern irrespective of the preferred route of DNA transfer both in vitro and in vivo.
  • anionic liposomes that better resemble cell-derived liposomes (in term of their electric charge) were also shown to mediate gene transfer, but suffer from poor encapsulation efficiency due to the large size and the negative charge of the uncondensed DNA. Improving encapsulation efficiency and protecting DNA from degradation was achieved by complexation of the DNA with cations or poly-cations that subsequently also significantly improved the transfection efficiencies.
  • MSC adult mesenchymal stem cells
  • HSC adult hematopoietic stem cells
  • endothelial cells accumulate at tumor microenvironments, when administered to tumor bearing animals.
  • MSC mesenchymal stem cells
  • HSC adult hematopoietic stem cells
  • endothelial cells accumulate at tumor microenvironments, when administered to tumor bearing animals.
  • MSC mesenchymal stem cells
  • HSC adult hematopoietic stem cells
  • endothelial cells endothelial cells
  • the homing mechanism motivated studies on the use of these cells as a targeted delivery vehicle for cancer therapy.
  • primary cells were isolated and transduced with different genes of interest, either anti-cancer or reporter genes.
  • the cells were transplanted to tumor bearing animals and their homing to the tumor microenvironment was demonstrated using the expressed reporter proteins. Tumor inhibition was achieved using the expressed anti-cancer proteins.
  • Liposomes which are derived from the cytoplasmatic membrane of mammalian cells, have been commonly used as a tool in the study of membranes and cellular mechanisms.
  • Cell derived liposomes (CDL or CDLs in plural) have been also investigated as a tool for cancer immunotherapy. In these studies, liposomes were prepared from the membranes of tumor cells and were used as adjuvant to evoke the immune system towards tumor antigens located on the liposome membrane.
  • cell derived liposomes have never been produced from stem cells, nor used as a delivery vehicle.
  • no CDL system has ever been developed as a targeting platform.
  • WESTERMAN LARRY E ET AL relates to "Liposomes composed of reconstituted membranes for induction of tumor-specific immunity", METHODS IN ENZYMOLOGY; ACADEMIC PRESS INC, SAN DIEGO, CA, US, (20030101), vol. 373, ISSN 0076-6879, PAGE 118 - 127 .
  • IMMORDINO MARIA LAURA ET AL relates to "Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential", INTERNATIONAL JOURNAL OF NANOMEDICINE, DOVE MEDICAL PRESS LTD, AUCKLAND, NZ, (20060101), vol. 1, no. 3, ISSN 1176-9114, PAGE 297 - 315 .
  • US 6120797 provides liposomes containing one or more N-acylated phosphatidylethanolamines, such liposomes being useful for localizing the delivery of bioactive agents to cells.
  • the present invention relates to a composition-of matter comprising a liposome attached to, or encapsulating a pharmaceutical agent, said liposome being composed of a whole cell membrane fraction, wherein said whole cell membrane fraction does not include lipids alone but also includes membrane proteins, wherein the liposome exhibits native membrane symmetry and expression of native markers, and wherein said cell is a human mesenchymal stem cell.
  • said cell membrane is genetically modified to express an exogenous protein.
  • said liposome encapsulates, or attached to a pharmaceutical agent.
  • composition-of-matter is non-immunogenic in a human subject.
  • a cell source of said whole cell membrane fraction comprises cells autologous to a host subject.
  • a cell source of said whole cell membrane fraction comprises cells non-autologous to a host subject.
  • said pharmaceutical agent is a diagnostic agent.
  • said liposome is attached to a synthetic polymer at an external surface thereof.
  • said liposome has a size range of 30-1000 nm.
  • said liposome is further composed of synthetic lipids.
  • the present invention relates also to a method of producing liposomes exhibiting native membrane symmetry and expression of native markers, the method comprising,
  • the method further comprises conjugating a synthetic polymer to said liposomes following step (c).
  • the present invention relates also to a method of encapsulating a pharmaceutical agent in a liposome, the method comprising making the liposomes according to the previous method and adding the pharmaceutical agent prior to the step of homogenizing.
  • the present invention relates also a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient the composition-of-matter and a pharmaceutically acceptable carrier.
  • the present invention in some embodiments thereof, relates to liposomal compositions and uses of same.
  • the present inventors have designed a novel delivery vehicle for targeted delivery of therapeutic and diagnostic agents into cells and tissues.
  • the delivery vehicle is liposome-based composed of a whole cell membrane fraction which comprises both natural lipids and proteins.
  • the delivery vehicles of the present invention may be formulated to be of low immunogenic potential, easily home to the target tissue and can be genetically modified to express therapeutic or targeting moieties.
  • the present inventors have generated liposomes composed of whole cell membranes of mesenchymal stem cells, which are well-known for their homing capacities as well as their immuno-suppressive abilities (i.e., their ability to reduce inflammation and suppress immune cells) and hypo-immunogenic features (i.e., stealth-like features that makes them less immunogenic and less recognizable as foreign matter when heterologously transplanted).
  • the liposomes exhibit the protein signature of mesenchymal stem cells and as such are expected to mediate similar immunosuppression and migratory properties as intact mesenchymal stem cells.
  • composition-of-matter comprising a liposome attached to, or encapsulating a pharmaceutical agent, said liposome being composed of a whole cell membrane fraction.
  • liposome refers to fully closed carrier molecules comprising a spherical lipid membrane which itself is in a liquid crystalline phase or a liquid gel phase, in which an entrapped liquid volume is contained. The two liquid phases are immiscible.
  • liposomes of the present disclosure also referred to herein as cell derived liposomes (CDLs), similar to membranes of cells, are in an entirely gel/liquid state and/or liquid crystal state and not in a solid state.
  • the liposomes of some embodiments of the present disclosure have an expected protein to lipid ration of about 0.8 w/w.
  • the protein content of hMSCc CDLs is about 0.8 mg/10 8 cells (as determined by Bradford assay).
  • the lipid content can be easily determined using the Stewart phospholipids assay. It is expected to be about 1 mg/ 10 8 cells.
  • Liposomes include niosomes, transfersomes, emulsions, foams, micelles, liquid crystals, dispersions, lamellar layers and the like.
  • the liposomes may be unilamellar or multilamellar.
  • the liposomes are unilamellar, as determined by Cryo-TEM.
  • the liposomes exhibit native membrane symmetry and expression of native markers.
  • Liposomes of the present disclosure are composed of a whole cell membrane fraction.
  • cell membrane or “cellular membrane” (which may be interchangeably used) refers to a biological membrane, which surrounds the cell or is an integral part of an organelle thereof (e.g., chloroplast, ER, golgi, mitochondrion, vacuole, nucleus and a lysosome).
  • organelle e.g., chloroplast, ER, golgi, mitochondrion, vacuole, nucleus and a lysosome.
  • the cell membrane refers to the plasma membrane.
  • plasma membrane presents proteins, which are associated with cell-to-cell interactions, as well as other recognition molecules, such as receptors that bind soluble ligands.
  • a whole cell membrane fraction refers to a fraction, which does not include lipids alone but also includes membrane proteins.
  • membrane proteins include, but are not limited to, integral proteins, transmembrane proteins, lipid anchored proteins and glycoproteins.
  • the whole cell membrane fraction also includes carbohydrates.
  • the cell is a eukaryotic cell [e.g., mammalian (such as human), plant, insect cell].
  • a eukaryotic cell e.g., mammalian (such as human), plant, insect cell.
  • the eukaryotic cell is a mammalian cell.
  • the cell can be a primary cell (i.e., non-immortalized and at times not cultured) or a cell-line.
  • a primary cell may be advantageous for clinical use where non-cultured cells are used in autologous or non-autologous (syngeneic allogeneic or xenogeneic) settings.
  • the eukaryotic cell is a stem cell.
  • stem cells refers to cells, which are capable of remaining in an undifferentiated state (e.g ., pluripotent or multipotent stem cells) for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (e.g ., fully differentiated cells).
  • the stem cell is a mesenchymal stem cell.
  • Mesenchymal stem cells are the formative pluripotent blast cells.
  • Mesenchymal stem cells give rise to one or more mesenchymal tissues (e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts, cardiac like cells) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines.
  • MSCs can be isolated from embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood, bone marrow, adipose and other tissues, although their abundance in the bone marrow far exceeds their abundance in other tissues.
  • MSCs have been shown to have immunosuppressive functions in various settings, including autoimmune diseases and transplantation, rendering liposomes generated therefrom ultimate tools in inflammatory and autoimmune settings.
  • MSCs mesenchymal stem cells
  • mesenchymal stem cell cultures are generated by diluting BM aspirates (usually 20 ml) with equal volumes of Hank's balanced salt solution (HBSS; GIBCO Laboratories, Grand Island, NY, USA) and layering the diluted cells over about 10 ml of a Ficoll column (Ficoll-Paque; Pharmacia, Piscataway, NJ, USA). Following 30 minutes of centrifugation at 2,500 x g, the mononuclear cell layer is removed from the interface and suspended in HBSS.
  • HBSS Hank's balanced salt solution
  • MSCs are then centrifuged at 1,500 x g for 15 minutes and resuspended in a complete medium (MEM, ⁇ medium without deoxyribonucleotides or ribonucleotides; GIBCO); 20 % fetal calf serum (FCS) derived from a lot selected for rapid growth of MSCs (Atlanta Biologicals, Norcross, GA); 100 units/ml penicillin (GIBCO), 100 ⁇ g/ml streptomycin (GIBCO); and 2 mM L-glutamine (GIBCO).
  • MEM complete medium
  • FCS fetal calf serum
  • Resuspended cells are plated in about 25 ml of medium in a 10 cm culture dish (Corning Glass Works, Corning, NY) and incubated at 37 °C with 5 % humidified CO 2 . Following 24 hours in culture, nonadherent cells are discarded, and the adherent cells are thoroughly washed twice with phosphate buffered saline (PBS). The medium is replaced with a fresh complete medium every 3 or 4 days for about 14 days. Adherent cells are then harvested with 0.25 % trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO) for 5 min at 37 °C, replated in a 6-cm plate and cultured for another 14 days.
  • Trypsin/EDTA GIBCO
  • Cells are then trypsinized and counted using a cell counting device such as for example, a hemocytometer (Hausser Scientific, Horsham, PA). Cultured cells are recovered by centrifugation and resuspended with 5 % DMSO and 30 % FCS at a concentration of 1 to 2 X 10 6 cells per ml. Aliquots of about 1 ml each are slowly frozen and stored in liquid nitrogen.
  • a cell counting device such as for example, a hemocytometer (Hausser Scientific, Horsham, PA).
  • MSC cultures can grow for about 50 population doublings and be expanded for about 2000 fold [ Colter DC., et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000 ].
  • MSC cultures utilized by the present disclosure preferably include three groups of cells, which are defined by their morphological features: small and agranular cells (referred to as RS-1, herein below), small and granular cells (referred to as RS-2, herein below) and large and moderately granular cells (referred to as mature MSCs, herein below).
  • RS-1 small and agranular cells
  • RS-2 small and granular cells
  • mature MSCs large and moderately granular cells
  • MSCs When MSCs are cultured under the culturing conditions of the present disclosure they exhibit negative staining for the hematopoietic stem cell markers CD34, CD11B, CD43 and CD45. A small fraction of cells (less than 10 %) are dimly positive for CD31 and/or CD38 markers. In addition, mature MSCs are dimly positive for the hematopoietic stem cell marker, CD117 (c-Kit), moderately positive for the osteogenic MSCs marker, Stro-1 [ Simmons, P. J. & Torok-Storb, B. (1991). Blood 78, 5562 ] and positive for the thymocytes and peripheral T lymphocytes marker, CD90 (Thy-1). On the other hand, the RS-1 cells are negative for the CD 117 and Stro1 markers and are dimly positive for the CD90 marker, and the RS-2 cells are negative for all of these markers.
  • Other cells which may be used as an effective source for whole cell membrane fraction include, but are not limited to, endothelial cells, hepatic cells, pancreatic cells, bone cells, chondrocytes, neuronal cells and the like.
  • the cells can be used native (i.e., not manipulated by genetic modification) or genetically modified to manipulate the membrane composition of the cell.
  • the advantage of genetic modification is in its increased efficiency. Essentially all (> 95 %) the CDLs generated from genetically modified cells express the gene-of-interest.
  • the gene-of-interest may be constitutively expressed on the cell source (by integration to the cells genome) or transiently expressed (episomal expression) such as to avoid hazardous implications of stable transfection agents (e.g., lentiviral and retroviral vectors).
  • the cells may be genetically modified to express a gene-of-interest (i.e., not naturally expressed in the native membrane but also in order to enhance the expression of endogenous proteins that are naturally expressed on the cell's membrane but in lower levels).
  • a gene-of-interest i.e., not naturally expressed in the native membrane but also in order to enhance the expression of endogenous proteins that are naturally expressed on the cell's membrane but in lower levels.
  • the gene-of-interest encodes a membrane protein.
  • the gene-of-interest may be a native membrane protein or modified to have a membrane localization signal and other motifs needed for membrane anchorage e.g., a transmembrane domain.
  • membrane proteins which may be heterologously (exogenously) expressed include, but are not limited to, a targeting protein (e.g., antibodies, receptors, membrane anchored ligands, decoys), a protein which affects the chemistry of the membrane (e.g., structural proteins, charged proteins), a diagnostic protein (e.g., an enzyme as described in length below) and a therapeutic protein (as described in length below).
  • a targeting protein e.g., antibodies, receptors, membrane anchored ligands, decoys
  • a protein which affects the chemistry of the membrane e.g., structural proteins, charged proteins
  • diagnostic protein e.g., an enzyme as described in length below
  • a therapeutic protein as described in length below
  • a targeting moiety includes a targeting protein such as an antibody, a receptor ligand and a non-proteinecious molecule such as carbohydrates, which binds cell surface or extra-cellular matrix markers.
  • a targeting protein such as an antibody, a receptor ligand and a non-proteinecious molecule such as carbohydrates, which binds cell surface or extra-cellular matrix markers.
  • PSMA prostate-specific membrane antigen
  • NAAG 3 conjugated to a transmembranal motif
  • LIME transmembranal motif
  • This may be achieved, by genetically engineering the cells (of which the CDLs are derived from) to express the chimeric or natural form of NAAG.
  • the expression plasmid encoding LIME is constructed by PCR and subsequent insertion of the corresponding fragment into pcDNA3.1 (Invitrogen).
  • the primers also have BamHI (5' primer and 3' primer) site extension to facilitate the subcloning.
  • the PCR product is digested with BamHI and inserted into corresponding sites in pcDNA3.1(+) (CLONTECH Laboratories, Inc.).
  • LIME-acetylaspartylglutamate the open reading frame can be inserted into plasmid coding LIME such that the NAAG is conjugated trough its N-terminus and maintains its C-terminus free to react with PSMA [i.e., LIME(C)-(N)NAAG-COOH].
  • expression plasmid encoding NAAG-LIME chimera can be constructed following the method described previously described for CD8-LIME chimera 5 . Fragments corresponding to NAAG and LIME transmembrane region were generated by PCR. Primers encoding the 3' sequences of the NAAG and the 5' sequences of the LIME fragment were designed to overlap, such that annealing of the two products yielded a hybrid template. From this template, the chimera is amplified using external primers containing XbaI sites. The NAAG-LIME chimera is inserted into pcDNA3.1(+).
  • surface marker refers to any chemical structure, which is specifically displayed at uniquely high density, and/or displayed in a unique configuration by a cell surface or extracellular matrix of the target cell/tissue.
  • the targeting moiety may be useful for targeting to tumor cells.
  • the intracellular environment of tumor cells is more alkaline compared to their immediate extracellular environment, which in turn is more acidic than the microenvironment found in the angiogenic blood vessels feeding the tumor.
  • many previous studies have shown that the surface charges of tumor cells is more negative compared to benign normal cells and even less invasive tumor cells. Accordingly, it may be useful to express membrane-bound enzymes and/or proteins, which will render the liposomes with a positive charge only in the acidic intermediate extracellular environment of the tumor.
  • any membranal protein with a pI of about 7.2-7.4 that falls between the high alkaline pH of the angiogenic blood vessels (pH>7.4) and the low acidic pH of the tumor immediate extracellular environment (pH ⁇ 7.2) can be used.
  • Such proteins can be specifically identified by cross referencing the RCSB Protein Data Bank (PDB) for human plasma membrane proteins.
  • the expected desirable pI (7.2-7.4) for those proteins can be calculated using the standard iterative algorithm 10, 11 that gives relatively precise results of pI calculations for raw protein sequences 12, 13 .
  • the algorithm is used in the Compute pI/Mw tool at the ExPASy server.
  • Such liposomes are expected to have negative or neutral charge in the alkaline microenvironment of the angiogenic tumor vessels and positive charge in the more acidic immediate extracellular environment of the tumor. Accordingly, this charge alteration will assist both liposomal extravasation, which is significantly enhanced for negative of neutral particles, and intra-tumor delivery which is more easily accomplished with positively charge particles 8, 14, 15 .
  • Diseases associated with a target cell/tissue specifically displaying a growth factor receptor/TAA surface marker which are amenable to treatment by the method of the present disclosure include, for example, some of the numerous diseases which specifically display growth factor receptors/TAAs, such as EGF receptor, platelet derived growth factor (PDGF) receptor, insulin like growth factor receptor, vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF) receptor, transferrin receptor, and folic acid receptor.
  • EGF receptor EGF receptor
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • transferrin receptor transferrin receptor
  • folic acid receptor folic acid receptor
  • Curr Opin Oncol 13, 506-13 Kuan et al., 2000. Brain Tumor Pathol. 2000;17:71-8 Malignant glioma, glioblastoma, head and neck, breast, colon, lung, prostate, kidney, ovary, brain, pancreas, bladder EGF receptor George, D., 2001. Semin Oncol 28, 27-33 Brain, prostate PDGF receptor Wang, Y., and Sun, Y., 2002. Curr Cancer Drug Targets 2, 191-207 Breast, lung, colon, prostate IGF receptor Rosen, L. S., 2001. Cancer J 7 Suppl 3, S120-8 ; Giles, F. J., 2001.
  • the ligand is an antibody or an antibody fragment, targeting antigens specific to a receptor on a target cell.
  • Antibodies can be monoclonal antibodies, polyclonal antibodies or antibody fragments, which are target specific.
  • the antibodies attached to the liposomes are anti-CD19, anti-CD20, or anti-CD22, for specific binding to a B-cell epitope. These antibodies or antibody fragments are typically derived from hybridomas that show positive reactivity toward the affected B-cells. It is contemplated that other antibodies or antibody fragments targeting any other cell in the body can be similarly used.
  • anti-CD19 antibodies are used to target liposome containing an entrapped agent to malignant B-cells. The antibody recognizes a unique epitope, the CD 19 surface antigen, on the B-cells.
  • an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of the gene-of-interest may be expressed in the cells from which membranes are later extracted.
  • the exogenous polynucleotide sequence may be a DNA or RNA sequence of the gene-of-interest.
  • the phrase "functional portion" as used herein refers to part of the encoded protein (i.e., a polypeptide), which exhibits functional properties of the enzyme such as binding to a substrate.
  • the functional portion of an antibody may be the variable region conferring specificity and additional/or alternatively the constant region, i.e., Fc, which may activate complement and induce cell killing.
  • Fc constant region
  • cells can be transfected with genes encoding one or more members from the GPCRs family (e.g., CCR5, CXCR4 etc.) that will render the liposomes targeted against abundant of cellular pathologies including auto-immune and viral diseases (e.g., HIV/AIDS).
  • a polynucleotide sequence encoding the gene-of-interest is preferably ligated into a nucleic acid construct suitable for eukaryotic cell expression.
  • a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • Constitutive promoters suitable for use for mammalian expression with the present disclosure are promoter sequences, which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with the present disclosure include for example the inducible promoter of the tetracycline-inducible promoter ( Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804 ).
  • the nucleic acid construct (also referred to herein as an "expression vector") of the present disclosure includes additional sequences, which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • the nucleic acid construct of the present disclosure typically includes a signal sequence for directing the translated polypeptide to the membrane and additionally a membrane anchor domain such as a transmembrane domain or a lipid based anchor (e.g., GPI).
  • a membrane anchor domain such as a transmembrane domain or a lipid based anchor (e.g., GPI).
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • the promoter utilized by the nucleic acid construct of the present disclosure is active in the specific cell population transformed.
  • cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [ Pinkert et al., (1987) Genes Dev. 1:268-277 ], lymphoid specific promoters [ Calame et al., (1988) Adv. Immunol. 43:235-275 ]; in particular promoters of T-cell receptors [ Winoto et al., (1989) EMBO J. 8:729-733 ] and immunoglobulins; [ Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [ Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477 ], pancreas-specific promoters [ Edlunch et al. (1985) Science 230:912-916 ] or mammary gland-specific promoters such as the milk whey promoter ( U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166 ).
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present disclosure include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983 .
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for the present disclosure include those derived from SV40.
  • the expression vector of the present disclosure may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of the present disclosure can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as lentiviruses and retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • cells, membranes, ghosts or CDLs may be chemically treated such as to present a protein, a saccharide, a synthetic polymer, a peptide or any combination of same.
  • Methods of modifying the membrane with a synthetic polymer are described herein below and in the examples section, which follows.
  • Such a chemical attachment may be effected at any stage from live cultured or suspended cells to produced CDLs.
  • the CDLs may be also chemically conjugated with folate that may further enhance their targeting and attachment to tumor cells, which are known to express higher levels of folate receptors compared to benign cells.
  • CDLs it is also possible to permanently modulate the CDLs to have a more positive surface charge by treating them with cations, salts or polycations (e.g., Polybrene®, polyethyleneimine and Poly-L-Lysine) rendering them more positive to better target the tumor angiogenic vasculature.
  • cations, salts or polycations e.g., Polybrene®, polyethyleneimine and Poly-L-Lysine
  • Non-native material can be also introduced to the surface of the CDLs by fusion (e.g., PEG or detergent induced) with other liposomes (e.g., cell-derived or synthetic) that may be comprised of well characterized lipids, proteins and additives.
  • fusion e.g., PEG or detergent induced
  • other liposomes e.g., cell-derived or synthetic
  • Such fusion, creating hybrid CDLs can be used to conjugate any moieties (e.g., targeting, therapeutic, diagnostic, stealth-rendering etc.) to the CDLs and to alter their surface properties. See Example 5 for further guidance on liposomal fusion.
  • Synthetic polymers are typically used to prevent or reduce coagulation, increase dispersion, reduce interaction with blood components, evade non-specific uptake by the mononuclear phagocytic system and prolong the particle circulation time to a large extent thus, rendering the liposomes with properties and features that are commonly referred to as stealth properties or long-circulating liposomes. Accordingly, the pH nano-environment at the particle surface may also be dependent upon the length of these molecules.
  • Polymers typically used as lipid modifiers include, without being limited thereto: polyethylene glycol (PEG), polysialic acid, polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), apolylactie- polyglycolic acid' polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyllydroxyetlyloxazolille, solyhydroxypryloxazoline, polyaspartarllide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • PEG polyethylene glycol
  • polysialic acid polylactic
  • polyglycolic acid also termed polyglycolide
  • the polymers may be employed as homopolymers or as block or random copolymers.
  • lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually distearylphosphatidylethanolamine (DSPE).
  • PE phosphatidyl ethanolamine
  • DSPE distearylphosphatidylethanolamine
  • a specific family of lipopolymers which may be employed by the disclosure include PEG-DSPE (with different lengths of PEG chains) in which the PEG polymer is linked to the lipid via a carbamate linkage and Polyethyleneglycol distearoylglycerol.
  • the PEG moiety headgroup preferably has a molecular weight from about 750 Da to about 20,000 Da. More preferably, the molecular weight is from about 750 Da to about 12, 000 Da and most preferably between about 1,000 Da to about 5,000 Da.
  • Two exemplary DSPE-PEG are those wherein PEG has a molecular weight of 2000 Da, and of 5000a designated herein DSPE-PEG(2000) (DSPE-PEG2k) and DSPE-PEG(5000) (DSPE-PEG5k).
  • Specific families of lipopolymers which may be also employed by the disclosure, include C8 and C16 mPEG Ceramides (with different lengths of PEG chains) in which the PEG-Ceramides contain ester linkages between the PEG and ceramide moieties that allow the compound to be easily metabolized.
  • the PEG moiety headgroup preferably has a molecular weight from about 750 Da to about 2,000 Da. More preferably, the molecular weight is about 2,000 Da.
  • CDLs may be also PEGylated by the two following described methods or their combination.
  • PEGylated CDLs will be prepared by detergent-dialysis incorporation of PEGylated lipids into the ghost cell membrane (prior to CDLs preparation).
  • direct PEGylation of the CDLs may be performed with monomethoxy-PEG activated by succinimidyl succinate, which has been proven to increase the trasnfection efficiency and reduce serum mediated inactivation of PEGylated lentiviral particles, used as gene transduction vectors 16 .
  • non-proteinaceous components e.g., synthetic polymers, carbohydrates and the like
  • a non-proteinaceous moiety may be covalently or non-covalently linked to, embedded or adsorbed onto the liposome using any linking or binding method and/or any suitable chemical linker known in the art.
  • the exact type and chemical nature of such crosslinkers and cross linking methods is preferably adapted to the type of affinity group used and the nature of the liposome. Methods for binding or adsorbing or linking the enzyme and/or targeting moiety are also well known in the art.
  • the enzyme and/or targeting moiety may be attached to a group at the interface via, but not limited to, polar groups such as amino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides.
  • the enzyme and/or targeting moiety may be attached via, but not limited to, active groups such as succinimidyl succinate, cyanuric chloride, tosyl activated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol, etc.
  • the enzyme and/or targeting moiety may be attached via, but not limited to, hydrophobic bonds (Van Der Waals) or electrostatic interactions that may or may not include cross-linking agents (e.g., bivalent anions, poly-anions, poly-cations etc.).
  • hydrophobic bonds Van Der Waals
  • electrostatic interactions may or may not include cross-linking agents (e.g., bivalent anions, poly-anions, poly-cations etc.).
  • the method may be practiced according to other well accepted protocols known in the art such as that of Boone, C.W., Ford, L.E., Bond, H.E., Stuart, D.C. & Lorenz, D. Isolation of plasma membrane fragments from HeLa cells. J Cell Biol 41, 378-392 (1969 ); and Westerman and Jensen Methods Enzymol. 2003;373:118-27 with or without modifications.
  • the term "ghosts” refers to a cell that all of its cytoplasmic contents and/or nucleolus were removed by cell lysis and/or membrane rapture so that only its outer cytoplasmatic/cell membrane remains;
  • liposomes of the present disclosure are made in a step-wise manner.
  • plasma membranes are isolated from cells (10 9 cells) primarily by using hypotonic treatment such that the cell ruptures and ghost cells are formed.
  • ghost cells can be formed using mild sonication, freeze-thaw, French-press, needle-passaging or solublization in detergent-containing solutions.
  • hypotonic treatment is effected in Tris-magnesium buffer (e.g., pH 7.4 or pH 8.6 at 4 °C, pH adjustment made with HCl). Cell swelling is monitored by phase-contrast microscopy.
  • the suspension is placed in a homogenizer. Typically, about 95% cell rupture is sufficient.
  • the membranes/ghosts are then placed in Sucrose (0.25 M or higher) for preservation.
  • the ghosts are placed in plastic tubes and centrifuged. A laminated pellet is produced in which the topmost lighter gray lamina consists only entirely of ghosts. However, the entire pellet is processed, to increase yields. Centrifugation (e.g., 3,000 rpm for 15 min at 4 °C) and washing (e.g., 20 volumes of Tris magnesium/TM-sucrose pH 7.4) may be repeated.
  • the ghost fraction is separated by floatation in a discontinuous sucrose density gradient. A small excess of supernatant is left over the washed pellet, which now contains ghosts, nuclei, and incompletely ruptured whole cells.
  • Additional 60% w/w sucrose in TM, pH 8.6 is added to the suspension to give a reading of 45 % sucrose on a refractometer.
  • all solutions contain TM pH 8.6. 15 ml of suspension are placed in SW-25.2 cellulose nitrate tubes and discontinuous gradient is formed over the suspension by adding 15 ml layers, respectively, of 40% and 35% w/w sucrose, and then adding 5 ml of TM-sucrose (0.25 M).
  • the material is now centrifuged at 20,000 rpm for 10 min, 4°C.
  • the nuclei sediment form a pellet
  • the incompletely ruptured whole cells are collect at the 40%-45% interface
  • the ghosts are collected at the 35%-40% interface.
  • the ghosts are collected and pooled.
  • the ghosts are homogenized such as by sonication which may be followed by extrusion.
  • a specific sonication protocol relates to 5 second sonication using an MSE sonicator with microprobe at an amplitude setting of 8 (Instrumentation Associates, N.Y.). This short period of sonication is enough to cause the plasma membrane of the ghosts to break up into cell derived liposomes (CDLs). Under these specific conditions organelle membranes are not disrupted and these are removed by centrifugation (3,000 rpm, 15 min 4 °C). Plasma membrane vesicles (CDLs) are then purified by sedimentation in a continuous sucrose density gradient.
  • Liposomes comprising one or more pharmaceutical agent of the present disclosure are preferably in the size range of 20-1000 nm e.g., 30-1000 nm, 0.02-1.0 ⁇ m, more preferably 0.05-1.0 ⁇ m, more preferably 0.07-0.5 ⁇ m and more preferably 0.1-0.3 ⁇ m.
  • An advantage of liposomes smaller or about 0.2 ⁇ m is that they can easily permeate through tumor vasculature (due to the EPR effect), they are not readily uptaken by macrophages and they can undergo filter sterilization.
  • Extrusion of liposomes through a commercially available polycarbonate membrane e.g., from Sterlitech, Washington
  • an asymmetric ceramic membrane e.g., Membralox
  • Pall Execia, France is an effective method for reducing liposome sizes to a relatively well defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller pore membranes (e.g., 400 nm, 100 nm and/or 50 nm pore size) to achieve a gradual reduction in liposome size and uniform distribution.
  • a pharmaceutical agent may be added to the reaction mixture such that the resultant liposomes encapsulate the pharmaceutical agent.
  • pharmaceutical agent refers to a therapeutic agent or diagnostic agent, which can be used to treat or diagnose a medical condition, respectively.
  • the composition comprising the pharmaceutical agent and the liposome is hypo or non-immunogenic especially when the cell source is a mesenchymal stem cell.
  • the liposome of the present disclosure may have a pharmaceutical agent adsorbed to a surface thereof or encapsulated therein either within the intra-liposomal polar phase or the lamellar non-polar lipid phase.
  • a the pharmaceutical agent may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from based on hydrophobic interactions (Van Der Waals bonds) or electrostatic interactions with or without the use of cross-linking agents (e.g. anions and poly-anions).
  • cross-linking agents e.g. anions and poly-anions
  • Hydrophobic and/or amphipathic pharmaceutical agent may be soulibilized, partially soulibilized or partitioned into the cells, ghosts or liposomal lipid membranes with or without the use of detergent and/or by detergent dialysis.
  • a pharmaceutical agent (or any other molecule) may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from based on covalent bonds with active groups.
  • a pharmaceutical agent may be attached, conjugated or adsorbed to surface of the liposomes, ghosts or the cells of which the liposomes derive from as a conjugate of an antibody or part of that specifically recognized a natural moiety found on the liposomes, ghosts or cells.
  • pharmaceutical agent may be adsorbed to the surface (inner or outer) of the liposomes via, but not limited to, polar groups such as amino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides.
  • the pharmaceutical agents may be adsorbed via, but not limited to, active groups such as succinimidyl succinate, cyanuric chloride, tosyl activated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol, etc.
  • active groups such as succinimidyl succinate, cyanuric chloride, tosyl activated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol, etc.
  • Entrapped in, adsorbed, expressed, conjugated, attached, and/or solubilzed on the liposomes' surface or membrane is a therapeutic agent for delivery to the target cells and/or tissues by one or more of ,but not limited to, the following mechanisms:
  • a variety of therapeutic agents can be entrapped in lipid vesicles, including water-soluble agents that can be stably encapsulated in the aqueous compartment of the liposome, lipophilic compounds that stably partition in the lipid phase of the vesicles, or agents that can be stably or transiently attached, conjugated, adsorbed or expressed on to the outer or inner surfaces of the liposomes, e.g., by electrostatic, covalent or hydrophobic interactions.
  • Exemplary water-soluble compounds include small molecules (i.e., less than 1000 Daltons) or large molecules (i.e., above 1000 Daltons); biomolecules (e.g. proteinaceous molecules, including, but not limited to, peptide, polypeptide, post-translationally modified protein, antibodies etc.) or a nucleic acid molecule (e.g. doublestranded DNA, single-stranded DNA, ds/ss RNA (e.g., siRNA, antisense, ribozymes), or triple helix nucleic acid molecules or chemicals.
  • Therapeutic agents may be natural products derived from any known organism (including, but not limited to, animals, plants, bacteria, fungi, protista, or viruses) or from a library of synthetic molecules. Therapeutic agents can be monomeric as well as polymeric compounds.
  • the therapeutic agent may be a protein, such as an enzyme which compensates for loss in activity or poor expression of an endogenous enzyme e.g., the enzyme hexosaminidase A, a shortage of which results in Tay-Sachs disease.
  • therapeutic agents which may be delivered across a blood barrier to the brain, eye, testis or mammary gland include, but are not limited to antibiotic agents, anti-neoplastic agents, anti-inflammatory agents, antiparasitic agents, antifungal agents, antimycobacterial agents, antiviral agents, anticoagulant agents, radiotherapeutic agents, chemotherapeutic agents, cytotoxic agents, cytostatic agents, vasodilating agents, antioxidants, analeptic agents, anti-convulsant agents, antihistamine agents, neurotrophic agents, psychotherapeutic agents, anxiolytic sedative agents, stimulant agents, sedative agents, analgesic agents, anesthetic agents, birth control agents, neurotransmitter agents, neurotransmitter analog agents, scavenging agents and fertility-enhancing agents.
  • the liposome-entrapped compound may also be a diagnostic agent such as an imaging or a contrast agent as indium and technetium, enzymes such as horseradish peroxidase and alkaline phosphatase, MRI contrast media containing gadolinium, X-ray contrast media containing iodine, ultrasonography contrast media such as CO 2 , europium derivatives, fluorescent substances such as carboxyfluorescein and illuminants such as N-methylacrydium derivatives.
  • a diagnostic agent such as an imaging or a contrast agent as indium and technetium
  • enzymes such as horseradish peroxidase and alkaline phosphatase
  • MRI contrast media containing gadolinium X-ray contrast media containing iodine
  • ultrasonography contrast media such as CO 2
  • europium derivatives fluorescent substances
  • carboxyfluorescein and illuminants such as N-methylacrydium derivatives.
  • the liposomes may be characterized for their size distribution, composition, concentration, zeta potential, electrical surface potential, surface (local) pH, protein to lipid ratio and therapeutic efficacy in vitro and in vivo.
  • Empty liposomes or liposomes comprising one or more pharmaceutical agent of the present disclosure are preferably in the size range of 30-3000-nm, more preferably 50-500 nm, more preferably 30-300 nm, more preferably 50-200 nm and more preferably 70-150 nm.
  • An advantage of liposomes smaller or about 100-nm is its ability to penetrate through very narrow blood vessels which is of great significance in diagnostic and treatment.
  • any method known in the art can be used to determine the size of the liposome.
  • a Nicomp Submicron Particle Sizer (model 370, Nicomp, Santa Barabara, Calif.) utilizing laser light scattering can be used.
  • Other methods of measuring liposome size include photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS), light obscuration methods (Coulter method, for example), rheology, or microscopy (light or electron).
  • LALLS low-angle laser light scattering
  • MALLS medium-angle laser light scattering
  • Coulter method for example
  • rheology light or electron
  • the preferred average effective particle size depends on factors such as the intended route of administration, formulation, solubility, toxicity and bioavailability of the compound.
  • liposomes of the present disclosure are characterized by a zeta potential of -20 to -15 mV without PEGylation and -15 to -10 mV with PEGylation.
  • liposomes of the present disclosure are advantageously used in the clinic.
  • a method of delivering a pharmaceutical agent comprising administering to a subject in need thereof the above-describe liposome, wherein the pharmaceutical agent is enclosed therein or adsorbed thereon, thereby delivering the pharmaceutical agent.
  • the cells are target cells and the liposomes contain a targeting moiety, either chemically conjugated, heterologously added, as described above, or natively presented in the membranes from which the liposome is comprised (e.g., as in MSCs, which migrate to tumor cells).
  • a targeting moiety either chemically conjugated, heterologously added, as described above, or natively presented in the membranes from which the liposome is comprised (e.g., as in MSCs, which migrate to tumor cells).
  • the cell source for the liposomes may be autologous or non-autologous (e.g., allogeneic, xenogeneic) to the subject.
  • the "target cell” referred to herein is a cell or a cluster of cells (of homogenous or heterogeneous population) and/or tissue to which a substance is to be delivered by using the liposome.
  • examples thereof include cancer cells, vascular endothelial cells of angiogenic cancer tissues, cancer stem cells, interstitial cells of cancer tissues, cells affected by genetic abnormality, cells infected by a pathogen and the like.
  • the "target molecule” may be any molecule presented the surface of the target cells or cells adjacent to the target cells. Another form of the target molecule includes molecules which are released from cells. Examples thereof includes extracellular matrix components, secretions or architectures of cancer cells or interstitial cells of cancer tissues, and specific examples thereof include tumor markers, structures between cells and the like.
  • Delivering can be for diagnostic reasons (e.g., the liposome includes a diagnostic agent) or for treating (i.e., as a drug delivery tool, delivering a therapeutic agent).
  • the liposomes may be administered to the subject per se, or as part of a pharmaceutical composition.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of the pharmaceutical composition is to facilitate administration of the active ingredients to the subject.
  • the term "active ingredient” refers to the therapeutic agent (with or without the liposome) accountable for the biological effect.
  • the liposome per se may have immunomodulatory function such as when prepared from membranes of MSCs or other immunomodulatory cells (e.g., immune B and T lymphocytes etc.).
  • the liposome per se may have a cytoxoic effect on the target cells as due to membrane fusion with target cells and consequent disruption to cell membrane, cytoskeleton and functions. In such a case measures are taken to include a targeting moiety such that the cytotoxic effect becomes specific.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered active ingredients.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to the pharmaceutical composition to further facilitate administration of an active ingredient of the present disclosure or to increase shelf-life stability.
  • excipients include calcium carbonate, calcium phosphate, various sugars and salts and types of starch, cellulose derivatives, gelatin, vegetable oils, EDTA, EGTA, Poly-L-Lysine, polyethyleneimine, Polybrene (hexadimethrine bromide), polyethylene glycols and other poly or single anions.
  • the pharmaceutical composition may advantageously take the form of foam, aerosol or a gel.
  • Suitable routes of administration include any of various suitable systemic and/or local routes of administration.
  • Suitable routes of administration may, for example, include the inhalation, oral, buccal, rectal, transmucosal, topical, transdermal, intradermal, transnasal, intestinal and/or parenteral routes; the intramuscular, subcutaneous and/or intramedullary injection routes; the intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, and/or intraocular injection routes, Catheterization with or without angio balloons; and/or the route of direct injection into a tissue region of the subject.
  • the pharmaceutical composition may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present disclosure can be delivered in the form of an aerosol/spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., a fluorochlorohydrocarbon such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane; carbon dioxide; or a volatile hydrocarbon such as butane, propane, isobutane, or mixtures thereof.
  • a suitable propellant e.g., a fluorochlorohydrocarbon such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane
  • carbon dioxide or a volatile hydrocarbon such as butane, propane, isobutane, or mixtures thereof.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pharmaceutical composition may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • a pharmaceutical composition for parenteral administration may include an aqueous solution of the active ingredients in water-soluble form.
  • suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredients may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the pharmaceutical composition may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the pharmaceutical composition should contain the active ingredients in an amount effective to achieve disease treatment.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture and in vivo assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1 ).
  • Dosage amount and interval may be adjusted individually to provide plasma or brain levels of the active ingredients which are sufficient to achieve the desired therapeutic effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of the composition to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present disclosure may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredients.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Human MSCs were purchased from Lonza® (Switzerland) and characterized using Giemsa staining and FACS analysis for human mesenchymal stem cells (hMSCs) typical cell surface markers. As seen from Figures 1A-C the cells appear to be positive for CD90, CD105, CD44, and CD29 and negative for CD133, CD31, CD34 and CD 144, as expected for hMSCs.
  • hMSCs were labeled by a red-fluorescent dye (DiI) while several other cell lines (including a prostate cancer cell-line - PC3) were labeled by a green fluorescent dye (DiO). Labeled cells were drop-wise seeded on tissue culture plates, incubated for 72 hrs and imaged using the Maestro in vivo Imager ( Figure 2 ). As seen, specific migration of hMSCs towards PC3 cancer cells was demonstrated while "avoiding" interaction with other cell-lines (BHK, Cf2Th, and COS-7).
  • Cell Derived Liposome preparation About 10 7 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl 2 ) pH 7.4 for 15min at 4°C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy).
  • TM buffer 0.01 M Tris, 0.001 M M MgCl 2
  • the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, CT) and centrifuged at 3,000 rpm for 15 min at 4°C.
  • the pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4°C.
  • the resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 ⁇ m and 0.1 ⁇ m (Osmonics Inc., Minnesota USA). The extruded liposomes were then centrifuged for 45 min at 150,000 g at 4°C. The supernatant was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.
  • Cell derived liposomes surface protein PEGylation (according to the methof of Croyle, M.A. et al., 2004 ) - About 10 7 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl 2 ) pH 7.4 for 15min at 4°C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy).
  • TM buffer 0.01 M Tris, 0.001 M MgCl 2
  • the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, CT) and centrifuged at 3,000 rpm for 15 min at 4°C.
  • the pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4°C.
  • the resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 ⁇ m and 0.1 ⁇ m (Osmonics Inc., Minnesota USA). The extruded liposomes were then centrifuged for 45 min at 150,000 g at 4°C. The supernatant was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.
  • the protein content on the liposome's surface was determined using the Bradford protein assay, referring to bovine serum albumin (BSA) as standard.
  • BSA bovine serum albumin
  • Succinimidyl succinate activated Monomethoxy-PEG was obtained from Sigma Chemicals (St. Louis, Mo.) and was added to the resuspended liposomes at a 10:1 ratio relative to the liposomes' protein content as previously determined by the Bradford assay. For example, 10 ⁇ g of Monomethoxy-PEG were added for each 1 ⁇ g of protein.
  • the conjugation reaction between the Monomethoxy-PEG and the liposomes was performed at 25°C with gentle agitation.
  • Liposomes were created form 2x10 7 hMSCs as previously described. Tosyl-activated paramagnetic Dynabeads® M-280 (invitrogen) were used as they were able to non-specifically and covalently bind any protein and/or liposomes conjugated with proteins and to be later analyzed by flow-cytometry. Using magnetic separation device (MACS, DynalTM Magnetic Particle Separator - Invitrogen), the beads were washed with the coupling buffer. To increase their ability to conjugate proteins, the beads were then further washed with 3M ammonium sulfate added to the coupling Buffer.
  • MCS Magnetic separation device
  • PEGylated Cell-Derived Liposomes are expected to be protected from opsonization and degradation, thus, having stealth properties and longer circulation time in vivo. Also, PEGylation may reduce the risk of non-specific binding and fusion of liposomes as with non-target cells 17-19 .
  • DiI-labeled CDLs were prepared from hMSCs, which were previously incubated for 24 hrs with condition media derived from a prostate cancer cell-line (PC3) and from a non-human cell-line (BHK).
  • PC3 prostate cancer cell-line
  • BHK non-human cell-line
  • the resulting "conditioned" CDLs, as well as CDLs prepared from unconditioned hMSCs (control) were incubated with PC3 and BHK cells for 15 min, 1 hr and 3 hrs. Following incubation, cells were washed, harvested and analyzed by flow cytometry ( Figure 9 ).
  • Mediums and buffers - 1L 2YT medium was prepared from 16 gr BactoTM Tryptone (BD number 211705), 10 gr BactoTM Yeast Extract (DIFCO number 212750) and 5 gr NaCl (Chemically Pure).
  • Medium used for culturing in Petri dished contained 16 gr Agar Granulated (DIFCO number 214530) on top of the above components.
  • the medium was autoclaved for sterility.
  • PBSX10 was prepred from 2 gr KCl, 2.4 gr KH 2 PO 4 , 14.4 gr Na 2 HPO 4 ⁇ 7H 2 O and 80 gr NaCl. Volume was adjusted to 1L with DDW and the buffer was filter sterilized through 0.2 ⁇ m filter.
  • Plasmids, DNA, bacteria and antibiotics - GST-sTRAIL coding DNA was kindly supplied by Dr. Stanley Lipkowitz, Bethesda, MD in pGEX-2TK plasmid introduced into E. coli BL21 using Ampicillin 100 ⁇ g/ml as a selection agent.
  • Ampicillin stock was prepared from ampicillin Sodium Salt (Sigma number A9518) dissolved in Ultra Pure DDW (UP-water) to a final concentration of 100 mg/ml and filtered through 0.2 ⁇ m filters.
  • Ethyl Alcohol 99% Dehydrated (FRUTAROM number 2355516400); D(+)GLUCOSE (Sigma number G5146); IPTG (Ornat Biochemicals number INA-1758-1400); Complete Mini EDTA-Free Protease inhibitor cocktail tablets (Roche Applied Science number 04693159001); DTT - DL-Dithiothereitol solution (Sigma number 43816); GSH BEADS (GE Healthcare); Glutathione Sepharose 4B (10 ml, Danyel Biotech number 17-0756-01); and L-Glutathione-reduced (Sigma number G4251).
  • STEP 2 The "starter" culture was spun down at 1000 g for 15 min to remove the antibiotics. The supernatant was discarded and resuspended in 40 ml of fresh 2YT.
  • the resuspended 40 ml of the O/N preparation from step number 1 was added to 2L of 2YT in a 4L flask (alternatively add the resuspended pellet of 20 ml of the O/N preparation from step number 1 to two 2L flasks each containing 1L of 2YT).
  • the solution was incubated for 2-3 hours in a 37°C shaking incubator at 250 RPM. Measures are taken not incubate for more than 3 hours until O.D. is 2.5-3.0 (it is recommended to measure O.D. 595 after 2 hours).
  • STEP 3 Just before IPTG induction, PBS was added to a final concentration of 0.1X to maintain the pH of the culture. EtOH (99% Dehydrated) was added to a final concentration of 2% (40 ml in 2L culture) to increase the solubility of the protein. 10 ml/L of 0.5M Glucose was added as a carbon source to a final concentration of 5mM.
  • STEP 4 500 ⁇ M IPTG were added to the supplemented culture. The culture was incubated over night in a shaking incubator (250 RPM) at 20-25° C.
  • STEP 5 Bacteria was pelleted at 6,000 g for 10 min and the supernatant was discarded. All bacteria were resuspended in a 50 ml Falcon Tube using 40 ml PBS supplemented with 4 protease inhibitor tablets (Roche Applied Science) 1 tablet per 10 ml PBS.
  • STEP 6 Cells were lyzed by running the bacteria from step number 5 twice through a French Press cell disruption system. Alternatively, 10 ml aliquots in 50 ml tubes were sonicated on ice at 30% power by 4 bursts of 10 sec each. After Cell disruption, the following was added to each 40 ml of cell lysate: 0.1% Triton-X (40 ⁇ l of TritonX100), 1 mM MgCl 2 (40 ⁇ l of 1M stock MgCl 2 ) and 1 mM DTT (40 ⁇ l of 1M stock DTT). The solution was mixed thoroughly and incubated at RT for 15 min on a rocker or shaker.
  • Triton-X 40 ⁇ l of TritonX100
  • 1 mM MgCl 2 40 ⁇ l of 1M stock MgCl 2
  • 1 mM DTT 40 ⁇ l of 1M stock DTT
  • STEP 7 The bacterial lysate was spun down for 10 min at 16,900 g and 4 °C. Supernatants were aspirated and collected in 50 ml tubes.
  • STEP 8 Binding to GSH (Glutathione - Sepharose 4B Beads) - In a 15ml Falcon Tube, 3 ml of GSH Beads were washed three times with PBS. Collected supernatant was centrifuged again because of mass bead loss. The washed beads were added to the bacterial cell lysate from step number 7 and incubated with tumbling for 1 hour at 4°C.
  • GSH Glutathione - Sepharose 4B Beads
  • STEP 9 The bacterial cell lysate, containing the sepharose beads from step number 8, was spun down at 2000 RPM for 1min in a MULTI CENTRIFUGE CM 6M ELMI to separate the protein-conjugated beads from the cell-lysate. The supernatant was collected and was centrifuged again to pellet the remaining sepharose beads in the supernatant (that might have not pelleted during the first centrifugation). The pellet from both centrifugations, containing the sTRAIL-conjugated beads, was washed 5 times with 5 ml of PBS supplemented with 0.1% Triton-X100, 150 mM NaCl and 1 proteinase inhibitor tablets per 20 ml PBS.
  • STEP 10 Elution of GST-sTRAIL - The beads were spun down as before and the supernatant was aspirated. 3 ml of 50 mM Glutathione (pH 8.5) in 10 mM Tris-HCl and 100 mM NaCl were added. Each 3 ml was vortexed for 2 min and the protein was eluted into the supernatant. The supernatant was aspirated as before and the supernatant kept. The procedure of elution was repeated 3-4 times.
  • STEP 11 The protein was concentrated using Amicon Ultra-15 10K NMWLnumber UFC9010, giving a protein yield of about 5 mg/L culture. sTRAIL was produced at a final concentration of 0.2 mg/ml
  • sTRAIL entrapment -About 10 7 Cells were harvested and washed with PBS. Cells were then hypotonically treated by re-suspension in ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl 2 ) pH 7.4 for 15min at 4°C. Following hypotonic treatment, the cells were homogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes as confirmed by phase-contrast microscopy).
  • TM buffer 0.01 M Tris, 0.001 M M MgCl 2
  • the re-suspended pellet was then sonicated for 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics & Materials Inc., Newtown, CT) and centrifuged at 3,000 rpm for 15 min at 4°C.
  • the pellet of sonicated ghosts was then washed twice again with 0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension and centrifugation at 3,000 rpm for 15 min at 4°C.
  • sTRAIL was added to the suspended sonicated ghosts (in TM buffer pH 8.6) to a final concentration of 1 ⁇ g per 1 ml of ghost suspension.
  • the sTRAIL-containing resuspeded pellet of sonicated ghosts was manually extruded by 21 successive passages trough polycarbonate membranes with pore sizes of 0.4 ⁇ m and 0.1 ⁇ m (Osmonics Inc., Minnesota USA). The extruded liposomes containing sTRAIL were then centrifuged for 45 min at 150,000 g at 4°C. The supernatant containing excess non-encapsulated sTRAIL was discarded and the resulting liposomes pellet was resuspended with TM buffer pH 8.6.
  • TRAIL - tumor necrosis factor-related apoptosis-inducing agent is a type II transmembrane protein that induces apoptosis in tumor cells of diverse origins, while sparing most normal cells 20-24 . Delivery of both full length and truncated, secreted forms of TRAIL (sTRAIL) were shown to induce apoptosis in a variety of cancer cells both in culture and in vivo 25, 26 . Our preliminary experiments with sTRAIL included its production and passive encapsulation within hMSCs CDLs at a final concentration of 1 ⁇ g/ml.
  • Various molecules can be introduced, conjugated or attached onto the surface of the CDLs by means of fusion between the CDLs and other liposomes (synthetic or cell-derive), thus creating - "Hybrid CDLs" .
  • a liposomal formulation made from synthetic well characterized lipids may be conjugated with a protein on its surface or may contain a desirable encapsulate. Then, by means of induced membrane fusion between the said synthetic liposomes and CDLs a hybrid CDL may be formed.
  • hybrid CDLs contain both lipids and proteins from the cell-membrane they derive from and the lipids and proteins that were originally formulated on the fused synthetic liposomes.
  • introduction of 'non-native' materials onto the Hybrid CDLs may be used to attach or conjugate any molecule or moieties related, but not limited to, liposomal targeting, therapeutic effect, diagnostic effect, stealth-rendering properties etc.
  • Such fusion may be also used to change the biochemical or chemophysical properties of the CDLs membranes by introduction of synthetic lipids, additives (e.g., cholesterol, ceramides) etc.
  • Such fusion may be also used to increase the encapsulation efficiency in the said CDLs. Since encapsulation in CDLs is mainly limited to passive encapsulation, fusion with synthetic liposomes that were actively loaded with high concentration of encapsulates may significantly improve the CDLs' encapsulation efficiency.
  • Synthetic liposomes of the said application can be produced by any method known in the art including, but not limited to, solvent evaporation, solvent replacement, detergent dialysis, extrusion, sonication, freeze-drying, reverse phase evaporation, ethanol/ether injection, agitation and/or any other form of mechanical homogenization.
  • Liposomes can be prepared from a variety of synthetic and naturally derived lipids and may or may not contain additional additives (e.g., cholesterol, ceramides etc.).
  • Methods for active encapsulation of matter in such synthetic liposomes which are mainly based on membrane pH gradient or active transporters, are also well known in the art and may be used to create synthetic liposomes with high encapsulation efficiency.
  • Fusion between CDLs and other liposomes to create "Hybrid CDLs" can be readily and easily accomplished by adding short chain free PEG ( ⁇ 200-500 Da) to the liposomes.
  • PEG short chain free PEG
  • the mechanism of PEG-induced vesicle fusion is believed to be related to the reduction of water activity and the dehydration of the lipid headgroups which consequently leads to vesicle coagulation and fusion. Fusion can also be artificially induced through electroporation in a process known as electrofusion. It is believed that this phenomenon results from the energetically active edges formed during electroporation, which can act as the local defect point to nucleate stalk growth between two bilayers.
  • Fusion can also be achieved by addition of detergents (usually under 2%) to the liposomal mixture (e.g., Cymal-5TM, 1-S-Octyl Beta-D-thioglucopyranoside etc.), incubation with mild agitation and consequent detergent dialysis.
  • detergents usually under 2%) to the liposomal mixture (e.g., Cymal-5TM, 1-S-Octyl Beta-D-thioglucopyranoside etc.), incubation with mild agitation and consequent detergent dialysis.
  • hMSCs were incubated for 24 prior to harvesting in medium composed of 50% conditioning media derived from PC3 cells. Cells were then harvested and sonicated ghosts and CDLs were prepared thereof by the method previously described. Sonicated ghosts from conditioned and unconditioned hMSCs (10 6 cells) were resuspended for analysis in 1 ml TM-buffer, pH 7.4. Cell-derived liposomes derived from 7x10 6 conditioned and unconditioned hMSCc were resuspended in 50 ⁇ L TM buffer, pH 8.6.
  • the samples were sent for proteomics analysis at the proteomics center of the TECHNION - Israel Institute of Technology. Briefly, the samples were digested by Trypsin and the resulting peptides were analyzed by LC-MS/MS. Peptide mix was fractionated by HPLC and electro-sprayed onto an ion-trap mass spectrometer (OrbitrapTM). Mass spectrometry was performed in order to analyze the peptides' mass to charge ratio spectra and to determine the proteins' mass. For additional analysis and identification, the peptides were further fragmented by collision induced dissociation (CID) and analyzed again. The peptides were identified by Sequest 3.31 software against the human part of the uniprot database. All protein results are given as Uniport Accession Numbers. The following values were determined for each protein/accession number:
  • the hundreds of proteins that were identified on one or more of the four samples can be divided into 4 distinct groups:

Claims (15)

  1. Materialienzusammensetzung, die ein Liposom umfasst, das an einem pharmazeutischen Mittel angebunden ist oder dieses umkapselt, wobei das Liposom aus einer Ganzzellenmembranfraktion zusammengesetzt ist, wobei die Ganzzellenmembranfraktion nicht nur Lipide umfasst, sondern auch Membranproteine umfasst, wobei das Liposom eine native Membramsymmetrie und Expression von nativen Markern zeigt, und wobei die Zelle eine humane mesenchymale Stammzelle ist.
  2. Materialienzusammensetzung nach Anspruch 1, wobei die Zellmembran genetisch modifiziert ist, um ein exogenes Protein zu exprimieren.
  3. Materialienzusammensetzung nach Anspruch 2, wobei das Liposom ein pharmazeutisches Mittel verkapselt oder an diesem angebunden ist.
  4. Materialienzusammensetzung nach Anspruch 1 bis 3, die in einem humanen Individuum nicht immunogen ist.
  5. Materialienzusammensetzung nach Anspruch 1 bis 4, wobei eine Zellquelle der Ganzzellenmembranfraktion Zellen umfasst, die in Bezug auf ein Wirtsindividuum autolog sind.
  6. Materialienzusammensetzung nach Anspruch 1 bis 4, wobei eine Zellquelle der Ganzzellenmembranfraktion Zellen umfasst, die in Bezug auf ein Wirtsindividuum nicht autolog sind.
  7. Materialienzusammensetzung nach Anspruch 1, wobei das pharmazeutische Mittel ein diagnostisches Mittel ist.
  8. Materialienzusammensetzung nach Anspruch 1 bis 7, wobei das Liposom an einem synthetischen Polymer an einer Außenoberfläche dieses angebunden ist.
  9. Materialienzusammensetzung nach Anspruch 1 bis 8, wobei das Liposom einen Größenbereich von 30 bis 1000 nm aufweist.
  10. Materialienzusammensetzung nach Anspruch 1 bis 9, wobei das Liposom ferner aus synthetischen Lipiden zusammengesetzt ist.
  11. Verfahren zum Herstellen von Liposomen, die eine native Membransymmetrie und Expression von nativen Markern zeigen, wobei das Verfahren umfasst:
    (a) Aussetzen von humanen mesenchymalen Stammzellen gegenüber hypotonischen Bedingungen, um gerissene Zellrupturen und/oder -schatten zu erhalten; und
    (b) Homogenisieren der gerissenen Zellmembranen und/oder -schatten, um Liposome zu erzeugen.
  12. Verfahren nach Anspruch 11, wobei das Homogenisieren bewirkt wird durch:
    (c) Beschallen der gerissenen Zellmembran und/oder -schatten; und optional
    (d) Extrudieren der gerissenen Membran und/oder -schatten durch eine Matrix mit vordefinierter Porosität.
  13. Verfahren nach Anspruch 11, das ferner das Konjugieren eines synthetischen Polymers an die Liposome nach Schritt (c) umfasst.
  14. Verfahren zum Verkapseln eines pharmazeutischen Mittels in einem Liposom, wobei das Verfahren das Herstellen der Liposome gemäß dem Verfahren nach Anspruch 11 und das Hinzufügen des pharmazeutischen Mittels vor dem Schritt des Homogenisierens umfasst.
  15. Pharmazeutische Zusammensetzung, die als Wirkstoff die Materialienzusammensetzung nach Anspruch 1 und einen pharmazeutisch annehmbaren Träger umfasst.
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IL130822A (en) 1996-10-15 2005-12-18 Elan Pharm Inc N-acyl phosphatidylethanolamine-mediated liposomaldrug delivery
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CN106619515A (zh) 2009-08-27 2017-05-10 工业研究与发展基金会有限公司 脂质体组合物及其用途

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US20120164214A1 (en) 2012-06-28
US20190224125A1 (en) 2019-07-25
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US20170224618A1 (en) 2017-08-10
US20210267892A1 (en) 2021-09-02
US11065203B2 (en) 2021-07-20
WO2011024172A2 (en) 2011-03-03
WO2011024172A3 (en) 2011-05-26
US9642817B2 (en) 2017-05-09
CN102596179A (zh) 2012-07-18
US10292933B2 (en) 2019-05-21

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